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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2020-03-18 to 2020-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
yes
Remarks:
No NOELR/LOELR was reported
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 12-15 November 2019 / Date of signature: 27 August 2020
Specific details on test material used for the study:
- Density (certified): 0,921 kg/dm3
- Water solubility: 1.055 µg/L (20 °C, pH 7.164) (determined at Noack Laboratorien GmbH, 181120TL/CWE18483)
- Storage conditions: 18-25°C, dark, in the tightly closed original container
Analytical monitoring:
yes
Details on sampling:
Samples of test media including the control group were taken from alternating test replicates on days -2, -1 and during the exposure on study days 0, 7, 8, 14, 21, 28, 34 and 35. Additional sampling of the test item group took place on day 22 and day 29. At each sampling day 3 samples were taken per (alternating) test replicate. For the sampling the test solutions were directly weighed into the sampling vials (50 per vial).

- Preparation of the samples: 50 mL of the test item concentrations and the control were added with approx. 10% sodium chloride and liquid extracted with 2.5 mL cyclohexane by manual agitation for 2 min (enrichment factor 20). Before analysis, the organic phase was separated and dried with a small quantity of sodium sulfate. If necessary, the samples were diluted with dilution factor 20 (0.05 mL sampled added to 0.95 mL cyclohexane). 0.1 mL of internal standard solution (undecanoic acid, derivative, at 0.5 mg/L) were added to 1 mL of each sample prior to analysis.

- Sample storage: All samples were immediately extracted and the extracts were stored at room temperature. Until the start of the analysis the prepared samples were stored on an autosampler.

- Evaluation: Quantification of the test item was calculated by peak area based on the external standard, using the internal standard for correction purpose.
Vehicle:
no
Details on test solutions:
A water accommodated fraction (WAF) was daily prepared because the test item is an UVCB substance with compounds of different water solubilities. This procedure is in accordance with the OECD guidance document No. 23 (2019) and ASTM D6081 (2019). Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Four water accommodated fractions (WAF) with a nominal loading of 10 mg/L were prepared per study day in 20 L glass aquaria (one WAF per test replicate). The WAFs were prepared about 72 ± 2 hour prior to the start of the flow-through system as well as 72 ± 2 hour prior to the following days during the course of the study. For the limit loading rate, an appropriate amount of the test item (the density of the test item of 0.921 kg/dm3 was taken into account) was placed by pipette onto the surface of the dilution water. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for about 72 ± 2 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for 15 minutes at room temperature. Then, the WAF was pumped by membrane piston pumps from the approximate bottom of the water body to the test aquaria. This procedure did not cause droplets by eye.
- Eluate: Dilution water
- Differential loading: The study was performed as a limit test with a nominal loading rate of 10.0 mg test item/L.
- Control: Dilution water (without test item)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The WAF was checked via laser beam (Tyndall effect) for undissolved test item and microscopically for dispersed droplets (phase-contrast microscope, magnification: 400 times). Neither undissolved test item nor dispersed droplets were observed during the course of the study.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM

- Common name: Danio rerio (zebrafish)

- Source: All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany).

- Maintenance of brood fish: A breeding stock of unexposed, mature zebrafish with an age of approx. 8 to 11 months was used for the egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1L water per fish.
Temperature = 25+/-2°C;
Dissolved oxygen concentration > 60% of air saturation value;
pH value = 6-8.5;
Photoperiod = 16h light and 8h dark cycle (2 transition periods, 30 minutes each);
Diffuse light (7-750 lux on water surface);
Food = Artemia salina nauplii, 48 hours old, at libitum; Daphnia magna, juvenile and adult daphnids, ad libitum; dry food sera vipan SERA, ad libitum.
No disease treatments were administered.

- Water: Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24h to remove chlorine.
Nominal water parameters:
Total hardness = 10-250 mg CaCO3/L
pH value = 6.0-8.5
Alkalinity = 0.6 mmol/L (recent measurement: 2020-01-21)
Acidity = 0.2 mmol/L (recent measurement: 2020-01-21)
Conductivity = 149 µS/cm (recent measurement: 2020-01-21)

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS

- Spawning: 15 – 35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days and thus not medically treated. About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless-steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After 1.5 hours the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded (less than 30%). About 250 eggs were taken and washed in dilution water. Eggs originated from 3 different spawnings. The eggs that were used to start the exposure were pooled and attributed randomized to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs).

- Fertilization check: Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully
covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers can be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2 cell stage were regarded as not fertilized and discarded. The fertilization rate was 87%.

- Introduction of eggs: Only fertilized eggs with more than 2 (4, 8, 16 and 32 cell blastomers) were placed directly in the test vessels. 20 eggs were introduced by random per replicate (corresponding to 80 eggs per treatment group). The eggs were transferred randomly into test vessels containing the respective exposure solutions. The distribution of eggs to the concentration groups was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contained the necessary number of eggs.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
35 d
Remarks on exposure duration:
35 days (30 days post hatch), depending on post-hatch day 0 (study day 5).
Hardness:
Total hardness of the test media was measured once per week from the control and the limit loading rate. The mean total hardness was 61 mg CaCO3/L and ranged from 56 to 64 mg CaCO3/L in the control.
Test temperature:
During the exposure the water temperature was recorded continuously (once per hour) with a data logger. The mean temperature was 26.1 °C. The minimum temperature was 24.6 °C and the maximum temperature was 27.4 °C. Except for a short period of about 3 hours the minimum measured temperature fell below the lower limit of 24.5 °C with 0.2 °C. The water temperature did not differ by more than ± 1.5 °C between successive days during the test.
The mean water temperature measured once per week from all replicates during the exposure period was 25.8 °C for the control. The minimum and maximum measured temperature for the control were 25.4 and 26.4 °C, respectively.
pH:
The mean pH-values in the control and test item group were 7.47 and 7.49 and ranged from 7.34 to 7.69 during the exposure period
Dissolved oxygen:
The dissolved oxygen concentrations in the control and the limit loading group were in the mean 88 and 90% of the air saturation value and ranged from 70 to 100% of the air saturation value during the exposure period, respectively.
Salinity:
Not applicable
Conductivity:
149 µS/cm (recent measurement: 2020-01-21)
Nominal and measured concentrations:
- Nominal loading rate of 10 mg/L.
- Measured test concentrations: see Tables in "Attached background material".
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria provided with mesh coated fittings allowing flow-through of test media (12.5 x 11 x 18 cm) were used. The volume of the test media was about 2.5 L.
- Type: Test vessels were covered with transparent lids.
- Aeration: A gentle aeration was applied for all test vessels.
- Type of flow-through (e.g. peristaltic or proportional diluter): A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through of the dilution water for the control group and the WAF for the test group, respectively. The accuracy of the water flow-through was checked prior to start of the exposure and three times per week thereafter.
- Renewal rate of test solution (frequency/flow rate): The mean flow rates through the test vessels of the control and test item group were 0.517 and 0.510 L/h, respectively.
- No. of fertilized eggs/embryos per vessel: 20 eggs were introduced by random per replicate (corresponding to 80 eggs per treatment group). For the whole study 160 eggs were inserted at the start of the exposure, whereof 154 vital hatched larvae were observed at the day of completion of hatch (study day 6 / post hatch day 1).
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Feeding of test fish: The feeding regime was ad libitum during the whole feeding period (study day 5 to 34). Feeding started 2 days after the beginning of hatch (on study day 5 (post-hatch day 0)). Larvae were fed with a starter food ST-1, as well as a suspension of the starter food ST-1 and fine milled brine shrimp
nauplii (2–8 times daily). 2 days after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (2 – 8 times daily).
- Biomass loading: the maximum biomass loading based on the 2.5 L volume of a single growth chamber was 431.6 mg/L. The biomass loading rate based upon a flow of 12.5 L/d through each single test aquaria was 86.3 mg/L/d.

TEST MEDIUM / WATER PARAMETERS
Same as used for Holding.
The water was filtered on activated charcoal to remove residual chlorine.

OTHER TEST CONDITIONS
- Light intensity: Light intensity was measured at the start of exposure on the surface of all test vessels and ranged from 191 to 361 lux (mean: 253 lux).
- Photoperiod: A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
All biological parameters were observed daily. Dead larvae/fish and coagulated or dead eggs were removed daily, if observed as described below.
- Hatching: The number of hatched larvae was determined daily until study day 7. All embryos hatched were counted as hatched, even if they had died directly afterwards. Eggs were only removed, when mortality of eggs/embryos was observed as specified below. On study day 5, 95% of the control larvae had hatched. Therefore, study day 5 was defined as post-hatch day 0 (= PHD 0). For
evaluation of hatch, all hatched larvae (even dead ones) were counted. The cumulative number of hatched larvae was used for evaluation.
- Mortality: criteria for mortality vary according to life stage.
For eggs/embryos: if fungus growth on eggs was observed, these eggs were removed and counted. Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance and change in coloration was checked daily. Mortality caused by absence of heartbeat was checked, if applicable. Dead eggs/embryos were discarded.
For larvae and juvenile fish: immobility and/or lack or reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded.
- Further effects: Abnormal appearance and behavior were also recorded daily. The number of larvae or fish showing abnormality of body form was recorded. Abnormal animals were only removed from the test vessels on dealth. Abnormalities, e.g. quiescence, hyperventilation, uncoordinated swimming, swim-up behavior, atypical quiescence and atypical feeding behavior were recorded by visually inspecting each replicate.
- Measurement of fish size: At the end of exposure (post-hatch day 30) the fish were euthanized in a Benzocaine solution and the individual total length of all survivors was measured to the nearest 0.5 mm with graph paper. The total length (from the tip of the snout to the tip of the longer lobe of the caudal fin) was measured.
- Measurement of fish wet weight: At the end of exposure (post-hatch day 30) all surviving fish were weighed on replicate basis to the nearest 0.1 mg. Fish were blotted on paper towels to remove excess moisture prior to weighing. The mean wet weight per animal was calculated from the number of surviving fish.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: No
Reference substance (positive control):
no
Remarks:
No reference item is recommended for this test according to the guideline.
Key result
Duration:
35 d
Dose descriptor:
EL10
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: hatching success, fry growth expressed as length and weight on PHD 30, post-hatch survival and overall survival
Remarks on result:
other: 35 days test period, equivalent to 30 days post-hatch
Details on results:
- Egg fertilization rate: The egg fertilization rate determined on study day 0 (start of the exposure) was 87%. Eggs were fully covered with the respective test solutions during fertilization check.

- Hatch and Definition of Post Hatch Day 0: The Hatch began on study day 3 in both test groups and was completed until study day 6. The last remaining egg was observed to be dead on study 7 in the limit loading rate and was removed
from the test vessel. Study day 5 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95% in both test groups (see Table 6.1.2/1 in "Any other information on results incl. tables"). No statistically significant differences were found between the control and the limit loading rate of 10 mg/L for all tested study days. No effects were observed in the maximum tested concentration 10 mg/L.

- Swim-up: Swim-up was observed for a 3 day period from study days 5 to 7 (see Table 6.1.2/2 in "Any other information on results incl. tables"). Newly hatched
fry began to swim up on study day 5 (PHD 0). On study day 7 (PHD 2), all surviving larvae had swum up. No statistical analysis of swim-up data was carried out.

- Fry survival (post-hatch survival): The post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥ 75%). The fry survival (post-hatch survival) at the end of the study was 89% in the control group and 84% in the limit loading rate. The post-hatch survival data of the test concentrations are given in Table 6.1.2/3 (in "Any other information on results incl. tables"). No statistically significant differences were found between the control group and the limit loading rate of the test item. No effects were observed in the maximum tested concentration 10 mg/L.

- Overall survival: The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0 was 14% in the control group and 20% in the limit loading rate. No decrease of the overall survival was detected in the limit loading rate of the test item (see Table 6.1.2/4 in "Any other information on results incl. tables"). No statistically significant differences were found between the control group and the limit loading rate of the test item. No effects were observed in the maximum tested concentration 10 mg/L.

- Fry growth: Fry growth, expressed as length and wet weight measurements, was measured on study day 35 (PHD 30) from all survivors (see Table 6.1.2/5 in "Any other information on results incl. tables"). No statistically significant differences were found for both parameters. No effects were observed in the maximum tested concentration 10 mg/L.

- Morphological and behavioral effects: No biologically significant morphological and behavioral effects were observed in the test item concentrations.

Table 6.1.2/1: Hatch / Hatching time of the control and the limit loading rate of the WAF of the test item





















































Nominal loading rate (mg/L)



Rep.



PHD -2



PHD -1



PHD 0



PHD 1



Study day 3



Study day 4



Study day 5



Study day 6



Cumulative hatching rate (%)



Control



1


2


3


4



20


0


5


20



70


70


80


90



90


100


90


100



90


100


100


100



Mean



11



78



95



98



10



1


2


3


4



30


5


35


35



100


85


90


80



100


100


90


90



100


100


90


90



Mean



25 (-)



89 (-)



95 (-)



95 (-)



(-) / (+) = No statistically / statistically significant difference from control group.


 


Table 6.1.2/2: Percent swim-up of hatched live fry of the control and the limit loading rate of the WAF of the test item





















































Nominal loading rate (mg/L)



Rep.



PHD -1



PHD 0



PHD 1



PHD 2



Study day 4



Study day 5



Study day 6



Study day 7



Swim up of hatched live fry (%)



Control



1


2


3


4



0


0


0


0



94


90


78


95



94


95


100


100



100


100


100


100



Mean



0



89



97



100



10



1


2


3


4



0


0


0


0



95


90


100


83



100


100


100


100



100


100


100


100



Mean



0



92



100



100



 


Table 6.1.2/3: Post-hatch survival on study day 35 (PHD30) of the control and the limit loading rate of the WAF of the test item












































Nominal loading rate (mg/L)



Rep.



Eggs introduced on study day 0



Cumulative number of hatched larvae



Vital larvae on study day 35 (PHD 30)



Post-hatch survival on study day 35 (%)



Control



1


2


3


4



20


20


20


20



18


20


20


20



16


18


17


18



89


90


85


90



Mean



20



19.5



17.3



88



10



1


2


3


4



20


20


20


20



20


20


18


18



14


19


16


15



70


95


89


83



Mean



20



19.0



16.0



84 (-)



(-) / (+) = No statistically / statistically significant difference from control group.


 


Table 6.1.2/4: Overall survival and overall mortality on study day 35 (PHD30) of the control and the limit loading rate of the WAF of the test item.







































Nominal loading rate (mg/L)



Rep.



Vital larvae on study day 35 (PHD 30)



Overall survival (%)



Overall mortality (%)



Control



1


2


3


4



16


18


17


18



80


90


85


90



20


10


15


10



Mean



17.3



86



14



10



1


2


3


4



14


19


16


15



70


95


80


75



30


5


20


25



Mean



16.0



80 (-)



20 (-)



(-) / (+) = No statistically / statistically significant difference from control group.


 


Table 6.1.2/5: Overview of fry growth: length and wet weight on study day 35 (PHD30) of the control and the limit loading rate of the WAF of the test item.

























































Nominal loading rate (mg/L)



Rep.



PHD 30 (End of exposure)



Mean total length per fish (mm)



Mean wet weight per fish (mg)



Control



1


2


3


4



18.6


18.7


18.2


19.2



54.2


50.1


51.1


59.9



Mean



18.7



53.8



± SD



0.355



3.84



CV (%)



1.90



7.13



10



1


2


3


4



19.4


19.2


19.3


18.4



56.4


53.2


54.3


51.0



Mean



19.1 (-)



53.7 (-)



± SD



0.397



1.93



CV (%)



2.08



3.59



(-) / (+) = No statistically / statistically significant difference from control group.

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant differences were detected between the control and the limit loading rate of the test item for all parameters (hatching success, fry growth expressed as length and weight on PHD 30, post-hatch survival and overall survival). No effects were observed at the maximum nominal loading rate 10 mg/L.
Executive summary:

The effects of the test substance on the early-life stage of fish (Danio rerio) were determined at the test facility according to OECD Guideline 210 from 2020-03-18 to 2020-04-22.


Since the test substance is a UVCB substance with compounds of different water solubility and due to the low aqueous solubility of the test substance, the test item solution was prepared as a water accommodated fraction (WAF). Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. The study was performed as a limit test with a nominal loading rate of 10.0 mg test item/L. The WAF was prepared and used for flow-through testing. The test was started by placing fertilized eggs into the test vessels and it lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio were exposed per control and limit loading rate (4 replicates with 20 eggs each).


The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.


On study day 5, 95% of the control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0). Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, posthatch survival and overall survival.


The concentration of the test substance was analytically verified via GC-MS/MS on study days -2, -1, 0; 7; 14; 21; 28; 34 and 35 in the test item loading rate and the control. Additional samples on day 22 and day 29 were analysed in the test item loading rate. As the active ingredient of the test item shows 9 significant peaks, a group evaluation of three major groups took place: group I with peaks 1 to 3, group II with peaks 4 to 6 and group III with peaks 7 to 9. The grouping was based on the retention times of the signals.


The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences. No statistically significant differences were detected between the control and the limit loading rate of the test item for all parameters (hatching success, fry growth expressed as length and weight on PHD 30, post-hatch survival and overall survival). No effects were observed at the maximum nominal loading rate 10 mg/L.

Description of key information

OECD Guideline 210, GLP, key study, validity 1:


35d-EL10 (Danio rerio) > 10 mg/L based on nominal loading rate.

Key value for chemical safety assessment

Additional information

One key study is available to assess the long-term toxicity of the registered substance to fish.

In this Fish, Early-life Stage Toxicity Test with Danio rerio (NOACK, 2020), the effects of the registered substance to the hatching success, fry growth, post-hatch survival and overall survival were determined at the test facility according to OECD Guideline 210 with GLP compliance. This study was performed as a limit test with a nominal loading rate of 10.0 mg test item/L. The WAF was prepared and used for flow-through testing. The test was started by placing fertilized eggs into the test vessels and it lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio were exposed per control and limit loading rate (4 replicates with 20 eggs each). On study day 5, 95% of the control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0). The concentration of the test substance was analytically verified via GC-MS/MS throughout the test.

No statistically significant differences were detected between the control and the limit loading rate of the test item for all parameters (hatching success, fry growth expressed as length and weight on PHD 30, post-hatch survival and overall survival). No effects were observed at the maximum nominal loading rate 10 mg/L. Therefore, the EL10 is > 10 mg/L based on the nominal loading rate of the test substance.